A fire resistant and water resistant enclosure for an operable computer digital data storage device is provided. The enclosure has walls made of gypsum or concrete and, in one embodiment, has two small ventilation openings which remain open during normal operation of the digital data storage device and during the presence of fire. This embodiment has no moving parts. In the presence of fire, air and superheated steam from inside the enclosure expand and flow outwardly through the ventilation passageways. As those gases flow outwardly, they simultaneously block or prevent the transfer of heat from the exterior fire inwardly through those passageways. A water resistant pouch or coating surrounding the storage device prevents damage from flood, water, steam generated by the insulation or smoke from passing through said passageways and damaging the storage device. Another embodiment is provided utilizing one or more movable hatches which, in the presence of fire, cannot completely close, allowing superheated steam to escape from the enclosure yet during normal operation allow for better ventilation and cooling. A method is also provided for protecting the data storage device wherein the walls of the enclosure are supersaturated with water. The walls are coated with a vapor barrier such as plastic skin, plastic container or wax to prevent evaporation of water. In the presence of fire, the vapor barrier melts and the excess water stored in the walls absorbs latent heat of vaporization and extends the amount of time over which the enclosure is able to protect the storage device from damage.

The present invention pertains to water and fire resistant enclosures for operable computer data storage devices. More particularly, by some estimates over 80% of the damage in a fire is actually caused by water exposure and not the fire! Extreme water pressure from fire hoses or damage from automatic sprinklers typically causes more damage than the fire itself. In addition, vast amounts of steam, superheated and generated by typical fire resistant insulations serve to also damage data storage devices if the steam is managed incorrectly. The present invention provides significant improvements in the cost and performance of these enclosures during normal operation as well as in the presence of a fire and/or flood.
A problem with the movable hatch and fan design of U.S. Pat. No. 7,211,742 is that when the enclosure walls are made of gypsum or concrete, in the presence of fire, the movable hatch (or hatches) completely seals the ventilation openings of the enclosure. The prior art describes multiple enclosures for data storage devices using water based fire resistant insulations in which the enclosures are completely sealed. Similar to the effect of a stove top pressure cooking device for cooking food, a completely sealed container combined with the water carried by and chemically bound inside the gypsum or concrete walls is turned to steam and becomes superheated and pressurized since it is unable to easily escape from the interior chamber of the enclosure. The superheated steam, useful in cooking turkey, can easily destroy a data storage device beyond repair and cause the loss of valuable data. Allowing said steam to escape by not completely sealing the enclosure can dramatically reduce the interior temperature of the enclosure by over 100° F. allowing for greatly improved chances when recovering data after a fire.
One aspect of the invention is to overcome the above stated problem by preventing the movable hatch (or hatches) from completely sealing the ventilation opening in the presence of fire. This aspect is useful for relatively high power (20 to 2000 watts) data storage devices. The movable hatch concept allows a relatively large opening (2% or more than the external surface area of the enclosure) for the computer data storage device to allow for maximum airflow during normal operations for sufficient cooling. When exposed to high ambient temperatures over 200° F., such as in a fire, the movable hatches would nearly completely close to protect from exterior heat. During the fire though, a critical difference from the prior art would be movable hatches that are designed to not completely close but allow a slight opening of 1% or less of the total external surface area of said enclosure for superheated steam to escape resulting in a dramatically cooler inner temperature than a completely sealed enclosure. The slight, yet critical, opening would allow the pressure inside the enclosure to equalize with the outer atmospheric pressure. From physics, the Combined Gas Law dictates [(P1V1)/T1=(P2V2)/T2], a drop in pressure from one condition to the next with no change in volume would result in a proportional drop in temperature (Kelvins).
When comparing a data storage device with and without the small steam exhaust opening, test results have shown that the interior temperature can be decreased over 100° F. with the addition of the partially closed ventilation passageway in a fire! This dramatic improvement is critical to the survivability of electronics as when exposed to over 300°, typical active data storage media such as hard disk drives can quickly degrade and could become unrecoverable resulting in the loss of irreplaceable data.
A critical issue arises in a design with hatches that never completely seal the inner cavity. As previously mentioned, water damage caused by fire fighters and automatic sprinklers can result in more damage than the fire itself. Water, in a flood, can obviously penetrate an enclosure that never seals. A critical aspect of this invention provides a water resistant barrier around the data storage device which is completely contained inside the fire resistant enclosure. As will be shown, this critical difference enables the invention to survive water damage from multiple sources including: fire hose spray, automatic sprinklers, complete submersion and water given off as steam from the fire resistant insulation.
Another aspect of the invention is to overcome the same problem by utilizing a hatchless design. The hatchless design could be used for relatively low power devices (5-20 watts) in which during normal operation, a sufficient amount of air can be transferred through the enclosure for cooling the computer storage device contained within. It is significant to note that this embodiment of the invention has no moving parts! In the presence of fire, the insulation and chemically bound water is released through the small ventilation passageway to conduct steam outwardly before the steam becomes superheated to dangerous temperatures. The ventilation passageway is sufficiently small to prevent the transfer of excessive heat from said fire therethrough.
A further aspect of the present invention is to increase the amount of water carried by the gypsum (or concrete) to prolong the time period during which the enclosure maintains a sufficiently low interior temperature to protect the data storage device. The walls are either supersaturated or supercharged with water according to the invention. Various techniques are also utilized to prevent the supersaturated walls from losing water to ordinary evaporation. The water is critical to successful operation of the enclosure, since it can dissipate enormous amounts of heat (latent heat of vaporization of water is equal to 2260 kJ/kg) as it is converted to steam. The amount of water dissipated during a fire is proportional to the duration of protection possible by the enclosure. Assuming the gypsum or concrete is supersaturated with twice the amount of water, the enclosure has twice the cooling power and in some cases can resist the presence of fire approximately twice as long!!
The present invention relates generally to apparatus for protecting an operable computer digital data storage device from damage and loss of data resulting from fire or water exposure. More particularly, the present invention provides a compact, low cost, fire and water resistant enclosure for an operable computer digital data storage device. Typical computer digital data storage devices include computer hard disk drives, optical disk drives, solid state memory devices, tape drives, computers, or any other device which can actively read and write digital data with the intent of storing and retrieving computerized digital data. In the description below, reference is occasionally made simply to a hard disk drive as an example of a computer data storage device. It is to be understood that such a reference below is an example and not a limitation of the invention to hard drives.
Also, in the description below, reference is occasionally made simply to a fan as an example of a device to force airflow through an enclosure. There are multiple devices which actively move air known by many names such as fan, blower, air pump, etc. It is to be understood that a reference of fan below is an example and not a limitation of the types of devices to actively move air.
As digital data storage devices become able to store staggering amounts of data, the loss of a digital data storage device in disasters such as fire or flood becomes more and more catastrophic. For the home consumer, digital pictures, digital video and digitized music are becoming more and more common. By some estimates, digital storage requirements are growing at an average compounding rate of over 100% per year. According to some studies, over 90% of businesses close after experiencing catastrophic data loss. Smaller businesses and remote offices are especially vulnerable as they are typically without the resources to develop sophisticated disaster recovery plans. There is clearly a need to provide a compact, low cost, reliable fire and/or water resistant enclosure for operating digital data storage devices. The prior art contains numerous examples of enclosures that are more complicated, not waterproof, less reliable, more costly resulting in data loss when exposed to fire, floods or water damage from the act of fighting a fire.
The present invention provides an improved compact, low cost, fire and/or water resistant enclosure for an operable computer digital data storage device. The phrase "fire resistant enclosure," as used herein and in the claims, is hereby defined as an enclosure resistant to fire that occurs outside the enclosure. Fire resistance enclosures are designed to protect the contents inside the enclosure. The present invention differs from prior art "fire suppression enclosures" that are intended to suppress or to prevent the spread of fire that occurs within the enclosure, as for example in Blackmon et al, U.S. Pat. No. 6,548,753. Fire suppression enclosures are meant to protect the spread of fire to nearby objects outside the enclosure. As a result, fire suppression enclosures can be made of thin metal walls. On the other hand, fire resistant enclosures, such as the present invention, must include relatively thick, thermally insulating walls which incorporate insulation materials such as gypsum, ceramic fiber insulation or other possible fire resistant insulations. Enclosures made of thick, insulation filled walls filled with material such as gypsum present significantly different and more difficult design challenges than simple thin-walled metal enclosures due to the heat producing nature of active electronic storage devices.
The present invention includes several alternate embodiments, all of which are intended to provide low cost, reliable fire resistant and water resistant enclosures for operating hard drives and other computer data storage devices. The embodiments include, for example, hatchless enclosures with and without fans; water resistant enclosures with and without movable hatches; free convection enclosures with and without movable hatches.
The primary difference between the present invention and all known prior inventions is that the primary enclosure uses a water based insulation but is never completely sealed. Whether the enclosure has a hatch or is hatchless, a small opening is always present to allow for super heated interior steam from the insulation to escape during a fire.
One embodiment of the present invention avoids the use of movable hatches, such as shown and described in U.S. patent application Ser. No. 11/112,552, referenced above, which application is hereby included herein by reference. The hatchless embodiment of the present invention also avoids the requirement of any fire or smoke sensing apparatus. Elimination of those components results in a significantly lower cost device. The linchpin for the preferred embodiment is the discovery that vents of a sufficiently small size (1% or less of the external surface area of said enclosure) formed in a relatively thick water based fire resistant insulation such as a gypsum or concrete wall, for example, 1.5 inches thick, will prevent damage by fire while simultaneously allowing enough air ventilation in the absence of fire to cool the operating storage device! We have tested the concept with a prototype subjected to fire, with no loss of data and no serious damage to the hard drive storage device! Prior to the water based insulation releasing steam, we believe that as the internal air temperature starts to rise in the presence of fire, the internal air expands and flows outwardly through the small vent or vents. (The fan or blower that may or may not be present forces air through the vents loses power and stops operating at the outset of a fire.) The outward flow of expanding air counteracts the flow of external heat from the fire through the small vents into the internal chamber. The small vent or vents can be designed as tortuous or labyrinthian passages to reduce the amount of external heat from a fire that flows into the inner chamber. Examples are described below and shown in the drawings.
Our discovery is confirmed by the following calculation. Assuming constant atmospheric pressure, the internal volume of air will expand proportionally to the temperature rise in Kelvin (Tkelvin=Tcentigrade+273.15) according to the ideal gas theory and Combined Gas Law [(P1V1)/T1=(P2V2)/T2]. Assuming an initial temperature of 298 K (25° C.) and a final temperature of 373 K (100° C.), the internal chamber volume of air is expected to increase by approximately 25%. Therefore, it is believed that 25% of the volume of air in the inner chamber will slowly flow outwardly through the vent or vents as the temperature of the inner chamber gradually increases by 75° C. By sizing the inner chamber and vents appropriately, we have discovered that an insulated and vented chamber can be sufficiently fire resistant to prevent loss of data from most fires!! Additionally, as the temperature inside the enclosure reaches 373 K (100° C.) in a fire, the chemically bound water molecules in the water based insulation such as in gypsum or cement begin to release as steam providing a continuous outflow of gas throughout the fires duration as long as water is still present in the insulation.
We are unaware of any prior art fire resistant enclosures for active data storage devices wherein vents in the walls remain open in the absence of fire and in the presence of fire. The prior art does include the Olzak et al U.S. Pat. No. 6,153,720 which teaches an aircraft recorder. The enclosure for the recorder includes vents which are normally closed but become opened in the presence of fire; a phase changing, heat absorbing layer melts and drains through the vents. The vents allow equalization of pressure if the recorder falls into the ocean. The Olzak et al enclosure does not teach or suggest forced air ventilation (or any ventilation) for an operating digital data storage device wherein significant amounts of heat are generated. It is simply not usable for the purpose of the present invention.
Another embodiment of the invention includes a water resistant, smoke resistant and heat conductive "pouch" (covering, water vapor barrier or waterproof enclosure) which surrounds the data storage device. The "pouch" and data storage device are both positioned inside a fire resistant enclosure. Heat generated by the data storage device is conducted through the "pouch" (preferably metal) and is thereafter transferred to the exterior of the fire resistant enclosure by a variety of techniques. The "pouch" is fully submersible and protects the device and stored data in up to 30 feet of water and/or possible damage from smoke.
Prevention of smoke or water damage is a significant issue with any enclosure with openings that readily allow water or smoke to enter the inner chamber and damage the data storage device. The present invention provides a waterproof barrier around the data storage device that conducts heat generated by the device without interfering with its operation and/or ability to properly dissipate heat.
The prior art contains devices with complicated closure mechanisms to seal off water or smoke which rely on electrically driven water sensors and electromechanical actuators to seal off openings. Because the nature of water intrusion can be sudden and the speed of closure finite, one cannot be completely certain of preventing all water damage with a sensor and actuator architecture. The present invention, by surrounding the data storage device in a water resistant 'pouch' is inherently water proof to any exposure or flood while requiring no actuation or electrical power to protect against water damage!
Another problem with prior art water based fireproof insulation enclosures is that, in some instances, when the water carried in the water based fire resistant insulation such as gypsum or concrete is exposed to a fire, the interior chamber becomes fully saturated with water vapor raising the humidity to 100%. That humidity can damage the data storage device. The waterproof barrier of the present invention can prevent damage to the data storage device from the 100% humidity condition within the chamber.
The prior art includes water and fire resistant enclosures for paper documents (see Gelb et al U.S. Pat. No. 4,992,310); however, such enclosures are wholly unsuitable for an operating computer data storage device due to a lack of any features to provide cooling to the active electronics during normal use. Without such features, the internal temperature could rise to hundreds of degrees preventing the normal operation of typical electronic storage devices. The prior art also includes cooling jackets for data storage devices, but the cooling jackets are not water resistant (see Cheon U.S. published application 2004/0190255).
The prior art includes a relatively large enclosure for operable digital data storage devices, such as the Engler U.S. Pat. No. 6,158,833, which dissipates heat generated by the digital data storage device by conduction through the insulated walls of the container. The Engler design requires a relatively large enclosure and a low wattage devices within the enclosure since it does not provide any active or fan-driven cooling system. The present invention, in contrast, provides a compact enclosure a fraction of the size of the Engler enclosure for similar wattage devices. The present invention has shown to reduce the size, weight and cost of the enclosure by up to 90% while accommodating the same or greater cooling capability. The compact size of the present invention in one embodiment is achieved primarily because of a forced-air cooling system not present in the Engler device.
The prior art includes Pehrsson WO 89/06921, which provides a concrete wall enclosure with ventilation openings, a fan, and movable hatches. However, the hatches are designed to fully block and completely seal the openings in the event of fire which would result in data loss from superheated steam being unable to escape the completely sealed container. The present invention, as noted above, never fully closes the ventilation openings overcoming the issue of superheated steam. Pehrsson also does not provide any water resistant or smoke protection for the contents of the enclosure which could result in loss of data due to damage from elements common in almost all fires such as 100% humidity, smoke, fire sprinklers or fire hoses. Pehrsson also has no provisions for complete water immersion common in hurricanes, basements with broken water pipes or flooding common in many areas.
The prior art includes other digital data storage device enclosures with a complicated array of active sensors and expensive electromechanical device to disconnect and completely seal in the inner chamber from the external environment, such as Pihl et al U.S. Pat. No. 5,479,341. The Pihl patent teaches of an enclosure that has no active cooling during normal operation and does not inherently protect the data storage device in advance of a flood but instead relies on complicated and expensive sensors and actuators to sense and shut the enclosure before water damage can occur. In addition, the Pihl enclosure is designed to completely seal off the protected interior chamber which results in no escape route for the superheated steam and elevated temperatures and pressure during a fire. The Kikinis U.S. Pat. No. 5,623,597 utilizes a rather complex, passive heat exchanger with a rather large heat sink structure. That design requires a cumbersome insulation injection mechanism to fill the heat sink space when a threshold temperature is sensed. The present invention includes a "free convection" embodiment having a much simpler and more robust design than the prior art resulting in a design that is inherently fire resistant and water resistant with no moving parts and can cost up to 90% less than previous inventions.
The prior art also includes the Kishon et al published U.S. application No. U.S. 2004/0064631 dated Apr. 1, 2004. The Kishon et al device utilizes passive conduction of heat generated by the data storage device through screws extending from ambient air to the device cover (see paragraph [0021]). This technique is limited by the relatively low amount of heat transferable through the metal screws. The active, fan-driven cooling provided by the present invention achieves a much greater cooling capacity. In addition, the Kishon patent teaches of a completely sealed enclosure and gypsum insulation which would result in superheated temperatures during a fire.
The prior art also includes forced air cooling systems for operational digital data storage devices, but not used together with a compact, fire resistant and water resistant enclosure.
A primary object of the invention is to provide a fire resistant and/or water resistant enclosure for an operable computer digital data storage device which is robust in design, can accommodate higher heat producing electronics and inexpensive to produce.
A further object of the invention is to provide a water based fire resistant insulation such as a gypsum or concrete enclosure which may or may not utilize a fan and one or more vents to create either free or forced convection cooling and in which one or more movable hatches close in the presence of fire, but wherein the hatch closure does not fully seal the vent or vents, so that steam may be released through the partially sealed vent or vents.
Another object of the invention is to provide a gypsum enclosure wherein the gypsum walls are supersaturated or supercharged with water, and wherein said additional water becomes vaporized in the presence of fire and greatly prolongs the time period during which the enclosure protects the data storage device from heat damage.
A further object of the invention is to provide a fire resistant and water resistant enclosure for an operating data storage device having a hatchless enclosure which avoids the requirement of any fire, water or smoke sensing apparatus, and which has no moving parts. The operational digital data storage device would utilize vents of a sufficiently small size to prevent damage by fire while simultaneously allowing enough air ventilation to pass therethrough in the absence of fire to cool the operating storage device.
Another object of the invention is to provide a fire resistant enclosure for an active data storage device wherein vents in the walls of the enclosure remain open in the absence of fire and in the presence of fire.
A further object of the invention is to provide an enclosure for an operating computer data storage device wherein a heat conductive "pouch" surrounds the storage device, which is submersible and inherently protects the data storage device and data stored therein in advance of any potential flood or water exposure in up to 30 feet of fresh water or salt water.
Other objects and advantages of the invention will become apparent from the following description and drawings wherein:

Fire resistant and water resistant enclosure for operable computer digital data storage device


Real-time media optimization may be provided. First, a remote session may be established with a remote computing device. Then, during the remote session, non-real-time media data may be exchanged with the remote computing device over a server path. Moreover, real-time media data may be exchanged with the remote computing device over a media path during the remote session.

Videoconferencing uses telecommunications of audio and video to bring people at different sites together for a meeting. This can be as simple as a conversation between two people in private offices (point-to-point) or involve several sites (multi-point) with more than one person in large rooms at different sites. Besides the audio and visual transmission of meeting activities, videoconferencing can be used to share documents, computer-displayed information, and whiteboards.

This patent is subject to a terminal disclaimer.

Real-time media optimization over remoted sessions


Apparatus and methods are described for treatment of morbid obesity using minimally invasive techniques. The apparatus includes a system of components that may be used separately or in combination for effectively reducing stomach volume, bypassing a portion of the stomach and/or small intestines, reducing nutrient absorption in the stomach and/or small intestines and/or depositing minimally or undigested food farther than normal into the intestines, thereby stimulating intestinal responses. The components described include an artificial stoma device, a gastric sleeve device, an intestinal sleeve device and a combined gastrointestinal sleeve device.

Bariatrics is the field of medicine encompassing the study of overweight, its causes, prevention and treatment. Bariatric surgery is a treatment for morbid obesity that involves alteration of a patient's digestive tract to encourage weight loss and to help maintain normal weight. The following history of bariatric surgery is provided by the American Society for Bariatric Surgery.
JEJUNO-ILEAL BYPASS: Kremen and Linner's Jejuno-ileal Bypass: Bariatric surgery has continually evolved since its initial sporadic and tentative introduction in the 1950's. The first bariatric procedure to be preceded by animal studies and subsequently presented to a recognized surgical society and published in a peer-reviewed journal was that of Kremen and associates in 1954. (Kremen, Linner et al. 1954) The case, which they presented, was of a jejuno-ileal bypass (JIB). Jejuno-ileal bypass involved joining the upper small intestine to the lower part of the small intestine, bypassing a large segment of the small bowel, which is thus taken out of the nutrient absorptive circuit. In the discussion of the case, Philip Sandblom of Lund, Sweden, alluded to the fact that, two years previously, Victor Henriksson of Gothenberg, Sweden, had performed a similar procedure for morbid obesity. In this case the redundant small bowel was excised rather than bypassed. Subsequently it was discovered that Dr. Richard Varco of the University of Minnesota independently performed JIB at the University of Minnesota Hospitals around the same time as the operation of Kremen et al. Varco's case was unpublished and the patient record lost, so that the exact procedure date is unknown. (Buchwald and Rucker 1984)
JEJUNO-COLIC BYPASS: As part of an ongoing study of morbid obesity, Payne et al. reported results of ten patients in whom an end-to-side jejuno-colic shunt had been performed. (Payne, DeWind et al. 1963) In jejuno-colic shunt, the upper small bowel was joined even further down the intestinal tract, to the colon, with the idea of bypassing an even longer segment of the nutrient absorptive gastrointestinal tract. These patients had episodes of uncontrollable diarrhea, dehydration and electrolyte imbalance. Because of the problem with diarrhea, most of these surgeries were eventually taken down and converted to end-to-side jejuno-ileostomies. Payne and Dewind subsequently advised against jejunocolic anastomoses, instead recommending end-to-side jejuno-ileostomy anastomosing the first 14 inches of jejunum to the last 4 inches of ileum. (Payne and DeWind 1969)
PROBLEMS WITH JEJUNO-ILEAL BYPASS: (JIB) Two variants of jejunoileal anastomosis were developed, the end-to-side (Payne and DeWind 1969) and end-to end (Scott, Dean et al. 1973) anastomoses of the proximal jejunum to distal ileum. In both instances an extensive length of small intestine was bypassed, not excised, excluding it from the alimentary stream.
In both these variants a total of only about 35 cm (18″) of normally absorptive small intestine was retained in the absorptive stream, compared with the normal length of approximately 7 meters (twenty feet). In consequence, malabsorption of carbohydrate, protein, lipids, minerals and vitamins inevitably occur, Where the end-to-side technique was used, reflux of bowel content back up the defunctionalized small intestine allowed absorption of some of the refluxed material resulting in less weight loss initially and greater subsequent weight regain.
Bile is secreted by the liver, enters the upper small intestine by way of the bile duct, and is absorbed in the small intestine. Bile has an important role in fat digestion, emulsifying fat as the first stage in its digestion. Bypassing the major site of bile acid reabsorption in the small intestine therefore further reduces fat and fat soluble vitamin absorption. As a result, huge amounts of fatty acids, which are normally absorbed in the small intestine, enter the colon where they cause irritation of the colon wall and the secretion of excessive volumes of water and electrolytes, especially sodium and potassium, leading to diarrhea. This diarrhea is the major patient complaint and has characterized jejunoileal bypass in the minds of patient and physician alike since the procedure was introduced.
Bile salts help to keep cholesterol in solution in the bile. Following JIB, the bile salt pool is decreased as a consequence of reduced absorption in the small intestine and bile salt losses in the stool. The relative cholesterol concentration in gallbladder bile rises and cholesterol crystals precipitate in the gallbladder bile, forming a nidus for development of cholesterol gallstones in the gallbladder. Specific vitamin deficiencies also occur; Vitamin D and Calcium deficiencies lead to thinning of bone with bone pain and fractures as a result of osteoporosis and osteomalacia. Bypass of the terminal ileum, which is the specific site of Vitamin B12 absorption, leads to Vitamin B12 deficiency with a specific peripheral neuropathy. Vitamin A deficiency can induce night blindness. Calcium Oxalate renal stones occur commonly following JIB, along with increased colonic absorption of oxalate. The colonic absorption of oxalate has been attributed to:
1. Exposure of colonic mucosa to excessive bile salts and possibly bile acids, increasing colonic permeability to oxalate, or
2. Excessive quantities of fatty acids in the gut form soaps with calcium, reducing its availability to form insoluble calcium oxalate leading to the persistence of soluble and absorbable oxalate in the colon.
Patients with intestinal bypass develop diarrhea 4-6 times daily. The frequency of stooling varying directly with fat intake. There is a general tendency for stooling to diminish with time, as the short segment of small intestine remaining in the alimentary stream increases in size and thickness, developing its capacity to absorb calories and nutrients, thus producing improvement in the patients nutrition and counterbalancing the ongoing weight loss. This happy result does not occur in every patient, but approximately one third of those undergoing "Intestinal Bypass" have a relatively benign course. Unfortunately, even this group is at risk of significant late complications, many patients developing irreversible hepatic cirrhosis several years after the procedure.
JIB is the classic example of a malabsorptive weight loss procedure. Some modern procedures utilize a lesser degree of malabsorption combined with gastric restriction to induce and maintain weight loss. Any procedure involving malabsorption must be considered at risk to develop at least some of the malabsorptive complications exemplified by JIB. The multiple complications associated with JIB while considerably less severe than those associated with Jejunocolic anastomosis, were sufficiently distressing both to the patient and to the medical attendant to cause the procedure to fall into disrepute.
Listing of jejuno-ileal bypass complications:
Mineral and Electrolyte Imbalance:
Decreased serum sodium, potassium, magnesium and bicarbonate
Decreased sodium chloride
Osteoporosis and osteomalacia secondary to protein depletion, calcium and vitamin D loss, and acidosis
Protein Calorie Malnutrition:
Hair loss, anemia, edema, and vitamin depletion
Cholelithiasis
Enteric Complications:
Abdominal distension, irregular diarrhea, increased flatus, pneumatosis intestinalis, colonic pseudo-obstruction, bypass enteropathy, volvulus with mechanical small bowel obstruction
Extra-intestinal Manifestations:
Arthritis
Liver disease, occurs in at least 30%
Acute liver failure may occur in the postoperative period, and may lead to death acutely following surgery.
Steatosis, "alcoholic" type hepatitis, cirrhosis, occurs in 5%, progresses to cirrhosis and death in 1-2%
Erythema Nodosum, non-specific pustular dermatosis
Weber-Christian Syndrome
Renal Disease:
Hyperoxaluria, with oxalate stones or interstitial oxalate deposits, immune complex nephritis, "functional" renal failure.
Miscellaneous:
Peripheral neuropathy, pericarditis, pleuritis, hemolytic anemia, neutropenia, and thrombocytopenia
The multiple complications associated with JIB led to a search for alternative procedures, one of which was gastric bypass, a procedure that is described in detail later. In 1983 Griffen et al. reported a comprehensive series comparing the results of jejuno-ileal bypass with gastric bypass. 11 of 50 patients who underwent JIB required conversion to gastric bypass within 5 years, leading Griffen to abandon jejuno-ileal bypass. (Griffen, Bivins et al. 1983)
JIB can be summed up as having:
a. Good Weight Loss,
b. Malabsorption with multiple deficiencies,
c. Diarrhea.
As a consequence of all these complications, jejuno-ileal bypass is no longer a recommended Bariatric Surgical Procedure. Indeed, the current recommendation for anyone who has undergone JIB, and still has the operation intact, is to strongly consider having it taken down and converted to one of the gastric restrictive procedures.
BILIOPANCREATIC DIVERSION: A modern variant of the Jejuno-ileal Bypass (JIB) is Biliopancreatic Diversion (BPD), a procedure that differs from JIB in that no small intestine is defunctionalized and, consequently, liver problems are much less frequent. This procedure was developed by Professor Nicola Scopinaro, of the University of Genoa, Italy. (Scopinaro, Gianetta et al. 1996)
This procedure has two components. A limited gastrectomy results in reduction of oral intake, inducing weight loss, especially during the first postoperative year. The second component of the operation, construction of a long limb Roux-en-Y anastomosis with a short common "alimentary" channel of 50 cm length. This creates a significant malabsorptive component that acts to maintain weight loss long term. Dr. Scopinaro recently published long-term results of this operation, reporting 72% excess body weight loss maintained for 18 years. These are the best results, in terms of weight loss and duration of weight loss, reported in the bariatric surgical literature to this date.
From the patient's perspective, the great advantages of this operation are the ability to eat large quantities of food and still achieve excellent, long-term weight loss results. Disadvantages of the procedure are the association with loose stools, stomal ulcers, and foul smelling stools and flatus. The most serious potential complication is protein malnutrition, which is associated with hypoalbuminemia, anemia, edema, asthenia, alopecia, generally requires hospitalization and 2-3 weeks hyperalimentation. BPD patients need to take supplemental calcium and vitamins, particularly Vitamin D, lifelong. Because of this potential for significant complications, BPD patients require lifelong follow-up. In BPD patients who have received 200-300 cm alimentary limbs because of protein malnutrition concerns, the incidence of protein malnutrition fell dramatically to range from 0.8% to 2.3%.
In 1988, Hess, using a combination of Scopinaro's BPD and the duodenal switch described by DeMeester in 1987, developed a hybrid operation with the advantages of the BPD, but without some of the associated problems. The duodenal switch, originally designed for patients with bile reflux gastritis, consists of a suprapapillary Roux-en-Y duodeno-jejunostomy. This allows the first portion of the duodenum to remain in the alimentary stream thus reducing the incidence of stomal ulcer. When combined with a 70%-80% greater curvature gastrectomy (sleeve resection of the stomach) continuity of the gastric lesser curve is maintained while simultaneously reducing stomach volume. A long limb Roux-en-Y is then created. The efferent limb acts to decrease overall caloric absorption and the long biliopancreatic limb diverting bile from the alimentary contents, specifically to induce fat malabsorption. This technique, was first presented by Hess in 1992 and first published in a paper by Marceau, Biron et al. in 1993 is known as Biliopancreatic Diversion with Duodenal Switch (BPDDS). This procedure is claimed to essentially eliminate stomal ulcer and dumping syndrome.
BPD and its variants are the most major procedures performed for obesity and it follows that prospective patients who wish to consider BPD should seek out experienced surgeons with life-long follow up programs.
Listing of complications of biliopancreatic diversion:
Protein Malnutrition 15%
Incisional hernia 10%
Intestinal obstruction 1%
Acute biliopancreatic limb obstruction Stomal Ulcer 3.0%
Bone Demineralization: Pre-op 25%; at 1-2 yrs, 29%; at 3-5 yrs 53%; at 6-10 yrs 14%.
Hemorrhoids 4.3%
Acne 3.5%
Night Blindness 3%
Operative Mortality 0.4%-0.8% (1122 subjects, 1984-1993)
GASTRIC BYPASS: Gastric Bypass (RGB) was developed by Dr. Edward E. Mason, of the University of Iowa, based on the observation that females who had undergone partial gastrectomy for peptic ulcer disease, tended to remain underweight following the surgery, and that it was very difficult to achieve weight gain in this patient group. He therefore applied the principles of partial gastrectomy to obese females, finding that they did indeed lose weight. (Mason and Ito 1967) With the availability of surgical staples, he was able to create a partition across the upper stomach using staples, and did not require removal of any of the stomach. Subsequent modifications of the technique include a pouch of 50 ml or less, a gastroenterostomy stoma of 0.9 mm, use of the Roux-en-Y technique to avoid loop gastroenterostomy and the bile reflux that may ensue. Lengthening of the Roux limb to 100-150 cm to include a greater element of malabsorption and improve weight loss and the use of the retrocolic and retrogastric routing of the gastrojejunostomy to ease the technical difficulties of the procedure and improve long term weight loss results. Staple line failures have been found to occur many years after the procedure, in consequence surgeons have responded by use of techniques designed to prevent this. These include transection of the stomach, in which the staple line is divided and the cut ends oversewn. An alternative technique using superimposed staple rows is claimed to exert its effect by crushing the stomach tissue causing firm scarring along the staple line. Additionally, there have been attempts to stabilize the gastroenterostomy by the use of a prosthetic band, fashioned into a ring positioned just above the junction of gastric pouch and small intestine. Gastric Bypass has also stood the test of time, with one series of greater than 500 cases, followed for 14 years, maintaining 50% excess weight loss.
The complications of gastric bypass are much less severe than those of Intestinal Bypass, and most large series report complications in two phases, those that occur shortly after surgery, and those that take a longer time to develop. The most serious acute complications include leaks at the junction of stomach and small intestine. This dangerous complication usually requires that the patient be returned to surgery on an urgent basis, as does the rare acute gastric dilatation, which may arise spontaneously or secondary to a blockage occurring at the Y-shaped anastomosis (jejunojejunostomy). Then there are the complications to which any obese patient having surgery is prone, these including degrees of lung collapse (atelectasis), which occur because it is hard for the patient to breathe deeply when in pain. In consequence a great deal of attention is paid in the postoperative period to encouraging deep breathing and patient activity to try to minimize the problem. Blood clots affecting the legs are more common in overweight patients and carry the risk of breaking off and being carried to the lungs as a pulmonary embolus. This is the reason obese patients are usually anticoagulated before surgery with a low dose of Heparin or other anticoagulant. Wound infections and fluid collections are quite common in morbidly obese patients. These complications are often exacerbated by the presence five or six inches of fatty tissue outside the muscle layers of the abdomen.
Complications that occur later on after the incision is all healed include narrowing of the stoma (the junction between stomach pouch and intestine), which results from scar tissue development. Recall that this opening is made about 10 mm in diameter, not much wider than dime. With an opening this small, a very little scarring will squeeze the opening down to a degree that affects the patient's eating. Vomiting which comes on between the 4th and 12th week may well be due to this cause. The problem can be very simply dealt with by stretching the opening to the correct size, by "endoscopic balloon dilatation", which usually involves a single procedure on a day stay basis to correct the problem. Wound hernias occur in 5-10% and intestinal obstruction in 2% of patients an incidence similar to that following any general surgical abdominal procedure.
Another late problem that is fairly common, especially in menstruating women, after gastric bypass is anemia. Since the stomach is involved in iron and Vitamin B12 absorption, these may not be absorbed adequately following bypass. As a result anemia may develop. The patient feels tired and listless, and blood tests show low levels of hematocrit, hemoglobin, iron and Vitamin B12. The condition can be prevented and treated, if necessary, by taking extra iron and B12. Since the food stream bypasses the duodenum, the primary site of calcium absorption, the possibility of calcium deficiency exists, and all patients should take supplemental calcium to forestall this.
Dumping is often mentioned as a complication of gastric bypass, but it really is a side effect of the procedure caused by the way the intestine is hooked up. Dumping occurs when the patient eats refined sugar following gastric bypass, this causes symptoms of rapid heart beat, nausea, tremor and faint feeling, sometimes followed by diarrhea. Of course no one likes these feelings, especially patients who love sweets! The upshot is, of course, that sweet lovers avoid sweets after gastric bypass and this is a real help to them in their efforts to lose weight. It should be noted that a few surgeons, expert at endoscopic/laparoscopic surgery, are performing Gastric Bypass using laparoscopic techniques.
Listing of complications following gastric bypass:
Early:
1. Leak
2. Acute gastric dilatation
3. Roux-Y obstruction
4. Atelectasis
5. Wound Infection/seroma
Late:
1. Stomal Stenosis
2. Anemia
3. Vitamin B12 deficiency
4. Calcium deficiency/osteoporosis
Silastic Ring Gastric Bypass & Vertical banded gastric bypass (Fobi): The use of rings to control the stoma size, proven with Vertical Banded Gastroplasty, has led to their adoption by some surgeons as an addition to gastric bypass procedures, again to control the stoma size and prevent late stretching of the opening and, hopefully, improve the long term weight maintenance results. Both silastic rings and Marlex bands have been used. Usually the recommendation is for the ring circumference to be considerably larger than that used in primary obesity procedures, so that the limiting effect only comes into play after some degree of stretching of the pouch has occurred. The complications following silastic ring gastric bypass include the same complications as for gastric bypass, plus band erosion.
GASTROPLASTY: During World War II, the Russians, as part of their war effort, developed a series of surgical instruments to staple various body tissues together as a simple and rapid method of dealing with injuries. This concept was adapted and refined by American surgical instrument makers after the war, leading to the surgical stapling instruments in use today. These are capable of laying down as many as four parallel rows of staples, to create a partition, or the instrument comes with a knife blade which will cut between the newly placed staple rows, dividing and sealing the stapled tissues simultaneously. Other instruments place circular rows of staples to join two tubes end-to-end, which is very useful in connecting intestine together.
The early use of such stapling devices in obesity surgery involved removal of three staples from the row and firing the stapler across the top part of the stomach. This staples the two stomach walls together, except at the point where the three staples were removed, where a small gap remains. The idea being that food that the patient takes in is held up in the segment of stomach above the staple line causing the sensation of fullness. The food then empties slowly through the gap (stoma) into the stomach below the staple line where digestion takes place normally. Unfortunately, the muscular stomach wall has a tendency to stretch and the stoma enlarges. It soon became apparent that while patients lost weight for the first few months while the stoma was small, they soon stopped losing, and, indeed, frequently regained all they had lost. Surgeons tried to counter this by reinforcing the opening between the two compartments (Gomez 1981), however these techniques were only partially successful. The search for a better gastroplasty was pursued by Dr. Edward E. Mason, Professor of Surgery at the University of Iowa. (Mason 1982) He realized that the lesser curvature part of the stomach had the thickest wall and was therefore least likely to stretch, so he used a vertical segment of stomach along the lesser curvature for the pouch. Additionally, he was very meticulous in defining the size of the pouch, measuring it at surgery under a standard hydrostatic pressure, and has shown that best results follow the use of a very small pouch, holding only 14 cc saline at the time of surgery. The third modification that he made was to place a polypropylene band (Marlex Mesh) around the lower end of the vertical pouch, which acts as the stoma, to fix the size of the outlet of the pouch, preventing it from stretching. This is done by use of the circular stapling instrument to staple the front and back walls of the stomach together, cutting out a circular window to allow the polypropylene band to be placed around the lower end of the pouch. His extensive studies showed that the correct circumference of the band is 5.0 cm. The whole operation is called Vertical Banded Gastroplasty (VBG). Correctly performed this operation produces good weight loss results. It has the advantages of being a pure restrictive procedure with no malabsorption component and no dumping. Of course sweet eaters will have to avoid sweets on their own if they have this procedure. Similarly there are few complications associated with Vertical Banded Gastroplasty, because all food taken in is digested normally, and anemia is rare and Vitamin B12 deficiency is almost unknown. The patient does have to be very careful to chew food completely to avoid vomiting, and to avoid high calorie liquids such as regular sodas and ice cream that pass easily through the stoma. A surgical variant of the VBG is the Silastic Ring Vertical Gastroplasty (SRVG), which is functionally identical to VBG but uses a silastic ring to control the stoma size. It should be noted that a few surgeons, expert in minimal access surgery are performing gastroplasty using laparoscopic techniques.
Listing of complications following vertical banded gastroplasty:
Stenosis with persistent vomiting, if untreated, causing neurological damage
Ulcer
Incisional hernia
Wound Infection
Band erosion
Leakage (including post-operative leakage due to failure of the initial closure and late leakage due to staple line failure and/or failure of the stomach walls to heal/fuse together)
GASTRIC BANDING: Another way to limit food intake is to place a constricting ring completely around the top end (fundus) of the stomach, creating an hourglass effect. The ring is placed near the upper end of the stomach, just below the junction of stomach and esophagus. This idea of gastric banding has been around for quite a number of years, and was pursued in Europe and Scandinavia particularly. Initially, a readily available material such as arterial graft was used for the band. The results, however, were not as good as RGB or VBG and the concept has only become popular with the development of modern bands designed for the task and techniques to measure the size of the "stoma" created under the band and associated pressures. An ingenious variant, the inflatable band was developed by Dr. Kuzmak (Kuzmak, Yap et al. 1990) who devised a band lined with an inflatable balloon. This balloon was connected to a small reservoir that is placed under the skin of the abdomen, through which, the balloon can be inflated, thus reducing the size of the stoma, or deflated thus enlarging the stoma. Even more ingenious, has been the development of models that can be inserted laparoscopically, thus saving the patient the discomfort of a large incision. Since the hourglass configuration only constricts the upper stomach, with no malabsorptive effect, it acts as a pure restrictive operation. Like VBG, the favorable consequences are absence of anemia, dumping and malabsorption, while the disadvantages include the need for strict patient compliance. Long-term results of this device are not yet available, but logic would suggest they are likely to be comparable to VBG results with an unknown additional effect due to manipulation of the inflatable balloon. At the present time there are two devices on the world market. The LapBand manufactured by Bioenterics, Carpenteria, Calif. and the Obtech device produced in Sweden by Obtech Medical AG. Only the LapBand is freely available in the USA at this time, having completed U.S. trials and been approved for use by the FDA.
Listing of Complications Following Gastric Banding
Operative:
Splenic Injury
Esophageal Injury
Conversion to Open Procedure
Wound Infection
Late:
Band slippage
Reservoir deflation/leak
Failure to lose weight
Persistent vomiting
Acid reflux

Gastrointestinal sleeve device and methods for treatment of morbid obesity


Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present invention relates to the discovery, identification, and characterization of novel human polynucleotides encoding proteins that share sequence similarity with animal proteases. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that either lack or over express the disclosed genes, antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed sequences that can be used for diagnosis, drug screening, clinical trial monitoring and the treatment of physiological disorders.
Proteases are enzymes that cleave polypeptide sequences. Carboxypeptidases are proteases that hydrolyze the peptide bonds at the carboxy-terminal end of a chain of amino acids and have been identified in a wide variety of cell types and animals. Peptidases have been implicated in a wide variety of biological processes including, but not limited to, digestion, coagulation, diabetes, prostate cancer, gynecological disorders, neurological disorders, and obesity. Accordingly, peptidases represent significant targets for regulatory control of a variety of physiological processes and pathways.
The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human proteins, and the corresponding amino acid sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal proteases, and especially carboxypeptidases. As such, the described NHPs represent a new family of protease-related proteins with a range of homologues and orthologs that transcend phyla and a broad range of species.
The novel human nucleic acid sequences described herein, encode proteins/open reading frames (ORFs) of 47, 88, 247, 92, 437, and 350 amino acids in length (see SEQ ID NOS: 2, 4, 6, 8, 10, and 12 respectively).
The invention also encompasses agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof that compete with native NHP, peptides, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the described NHPs (e.g., antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or to enhance the expression of the described NHP sequences (e.g., expression constructs that place the described sequence under the control of a strong promoter system), and transgenic animals that express a NHP transgene, or xe2x80x9cknock-outsxe2x80x9d (which can be conditional) that do not express a functional NHP.
Further, the present invention also relates to processes of identifying compounds that modulate, i.e., act as agonists or antagonists, of NHP expression and/or NHP product activity that utilize purified preparations of the described NHPs and/or NHP product, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.
The Sequence Listing provides the sequences of 6 protease-related ORFs that encode the described NHP amino acid sequences.
The NHPs, described for the first time herein, are novel proteins that are expressed in, inter alia, human cell lines, and human brain, pituitary, spinal cord, thymus, spleen, lymph node, bone marrow, trachea, lung, kidney, prostate, testis, thyroid, adrenal gland, stomach, small intestine colon, skeletal muscle, uterus, mammary gland, bladder, and cervix cells. The described sequences were compiled from gene trapped cDNAs and a clone isolated from a human prostate cDNA library (Edge Biosystems, Gaithersburg, Md.). The present invention encompasses the nucleotides presented in the Sequence Listing, host cells expressing such nucleotides, the expression products of such nucleotides, and: (a) nucleotides that encode mammalian homologs of the described genes, including the specifically described NHPs, and the NHP products; (b) nucleotides that encode one or more portions of the NHPs that correspond to functional domains, and the polypeptide products specified by such nucleotide sequences, including but not limited to the novel regions of any active domain(s); (c) isolated nucleotides that encode mutant versions, engineered or naturally occurring, of the described NHPs in which all or a part of at least one domain is deleted or altered, and the polypeptide products specified by such nucleotide sequences, including but not limited to soluble proteins and peptides in which all or a portion of the signal sequence in deleted; (d) nucleotides that encode chimeric fusion proteins containing all or a portion of a coding region of an NHP, or one of its domains (e.g., a receptor/ligand binding domain, accessory protein/self-association domain, etc.) fused to another peptide or polypeptide; or (e) therapeutic or diagnostic derivatives of the described polynucleotides such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructs comprising a sequence first disclosed in the Sequence Listing.
As discussed above, the present invention includes: (a) the human DNA sequences presented in the Sequence Listing (and vectors comprising the same) and additionally contemplates any nucleotide sequence encoding a contiguous NHP open reading frame (ORF) that hybridizes to a complement of a DNA sequence presented in the Sequence Listing under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0.1% SDS at 68xc2x0 C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley and Sons, Inc., NY, at p. 2.10.3) and encodes a functionally equivalent gene product. Additionally contemplated are any nucleotide sequences that hybridize to the complement of the DNA sequence that encode and express an amino acid sequence presented in the Sequence Listing under moderately stringent conditions, e.g., washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et al., 1989, supra), yet still encode a functionally equivalent NHP product. Functional equivalents of a NHP include naturally occurring NHPs present in other species and mutant NHPs whether naturally occurring or engineered (by site directed mutagenesis, gene shuffling, directed evolution as described in, for example, U.S. Pat. No. 5,837,458). The invention also includes degenerate nucleic acid variants of the disclosed NHP polynucleotide sequences.
Additionally contemplated are polynucleotides encoding NHP ORFs, or their functional equivalents, encoded by polynucleotide sequences that are about 99, 95, 90, or about 85 percent similar to corresponding regions of SEQ ID NO:1 (as measured by BLAST sequence comparison analysis using, for example, the GCG sequence analysis package using default parameters).
The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the described NHP gene nucleotide sequences. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances where the nucleic acid molecules are deoxyoligonucleotides (xe2x80x9cDNA oligosxe2x80x9d), such molecules are generally about 16 to about 100 bases long, or about 20 to about 80, or about 34 to about 45 bases long, or any variation or combination of sizes represented therein that incorporate a contiguous region of sequence first disclosed in the Sequence Listing. Such oligonucleotides can be used in conjunction with the polymerase chain reaction (PCR) to screen libraries, isolate clones, and prepare cloning and sequencing templates, etc.
Alternatively, such NHP oligonucleotides can be used as hybridization probes for screening libraries, and assessing gene expression patterns (particularly using a micro array or high-throughput xe2x80x9cchipxe2x80x9d format). Additionally, a series of the described NHP oligonucleotide sequences, or the complements thereof, can be used to represent all or a portion of the described NHP sequences. The oligonucleotides, typically between about 16 to about 40 (or any whole number within the stated range) nucleotides in length may partially overlap each other and/or the NHP sequence may be represented using oligonucleotides that do not overlap. Accordingly, the described NHP polynucleotide sequences shall typically comprise at least about two or three distinct oligonucleotide sequences of at least about 18, and preferably about 25, nucleotides in length that are each first disclosed in the described Sequence Listing. Such oligonucleotide sequences may begin at any nucleotide present within a sequence in the Sequence Listing and proceed in either a sense (5xe2x80x2-to-3xe2x80x2) orientation vis-a-vis the described sequence or in an antisense orientation.
For oligonucleotide probes, highly stringent conditions may refer, e.g., to washing in 6xc3x97SSC/0.05% sodium pyrophosphate at 37xc2x0 C. (for 14-base oligos), 48xc2x0 C. (for 17-base oligos), 55xc2x0 C. (for 20-base oligos), and 60xc2x0 C. (for 23-base oligos). These nucleic acid molecules may encode or act as NHP sequence antisense molecules, useful, for example, in NHP sequence regulation (for and/or as antisense primers in amplification reactions of NHP nucleic acid sequences). With respect to NHP sequence regulation, such techniques can be used to regulate biological functions. Further, such sequences may be used as part of ribozyme and/or triple helix sequences that are also useful for NHP sequence regulation.
Inhibitory antisense or double stranded oligonucleotides can additionally comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5xe2x80x2-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide can also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide will comprise at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an xcex1-anomeric oligonucleotide. An xcex1-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual xcex2-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2xe2x80x2-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be used to disrupt the expression and function of a targeted NHP.
Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and periodic updates thereof), Cold Spring Harbor Press, NY; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY.
Alternatively, suitably labeled NHP nucleotide probes can be used to screen a human genomic library using appropriately stringent conditions or by PCR. The identification and characterization of human genomic clones is helpful for identifying polymorphisms (including, but not limited to, nucleotide repeats, microsatellite alleles, single nucleotide polymorphisms, or coding single nucleotide polymorphisms), determining the genomic structure of a given locus/allele, and designing diagnostic tests. For example, sequences derived from regions adjacent to the intron/exon boundaries of the human gene can be used to design primers for use in amplification assays to detect mutations within the exons, introns, splice sites (e.g., splice acceptor and/or donor sites), etc., that can be used in diagnostics and pharmacogenomics.
Further, a NHP sequence homolog can be isolated from nucleic acid from an organism of interest by performing PCR using two degenerate or xe2x80x9cwobblexe2x80x9d oligonucleotide primer pools designed on the basis of amino acid sequences within the NHP products disclosed herein. The template for the reaction may be total RNA, mRNA, and/or cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express an allele of a NHP sequence.
The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequence of the desired NHP gene. The PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment can be used to isolate genomic clones via the screening of a genomic library.
PCR technology can also be used to isolate full length cDNA sequences. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express a NHP sequence, such as, for example, testis tissue). A reverse transcription (RT) reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5xe2x80x2 end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be xe2x80x9ctailedxe2x80x9d using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a complementary primer. Thus, cDNA sequences upstream of the amplified fragment can be isolated. For a review of cloning strategies that can be used, see e.g., Sambrook et al., 1989, supra.
A cDNA encoding a mutant NHP sequence can be isolated, for example, by using PCR. In this case, the first cDNA strand can be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying a mutant NHP allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5xe2x80x2 end of the normal gene. Using these two primers, the product is then amplified via PCR, optionally cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant NHP allele to that of a corresponding normal NHP allele, the mutation(s) responsible for the loss or alteration of function of the mutant NHP sequence product can be ascertained.
Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant NHP allele (e.g., a person manifesting a NHP-associated phenotype such as, for example, obesity, high blood pressure, etc.), or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant NHP allele. A normal NHP sequence, or any suitable fragment thereof, can then be labeled and used as a probe to identify the corresponding mutant NHP allele in such libraries. Clones containing mutant NHP coding sequences can then be purified and subjected to sequence analysis according to methods well known to those skilled in the art.
Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant NHP allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against a normal NHP product, as described below. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, xe2x80x9cAntibodies: A Laboratory Manualxe2x80x9d, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)
Additionally, screening can be accomplished using labeled NHP fusion proteins, such as, for example, alkaline phosphatase -NHP or NHP-alkaline phosphatase fusion proteins. In cases where a NHP mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), polyclonal antibodies to a NHP are likely to cross-react with a corresponding mutant NHP sequence product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known in the art.
The invention also encompasses (a) DNA vectors that contain any of the foregoing NHP coding sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences (for example, baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporated by reference); (c) genetically engineered host cells that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell; and (d) genetically engineered host cells that express an endogenous NHP sequence under the control of an exogenously introduced regulatory element (i.e., gene activation). As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the human cytomegalovirus (hCMV) immediate early gene, regulatable, viral elements (particularly retroviral LTR promoters), the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase (PGK), the promoters of acid phosphatase, and the promoters of the yeast xcex1-mating factors.
The present invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of the NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP sequence (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote the expression of a NHP (e.g., expression constructs in which NHP coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.).
The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body. The use of engineered host cells and/or animals may offer an advantage in that such systems allow not only for the identification of compounds that bind to an endogenous receptor/ligand of a NHP, but can also identify compounds that trigger NHP-mediated activity.
Finally, the NHP products can be used as therapeutics. For example, soluble versions or derivatives of a NHP, or peptides/domains corresponding a NHP, NHP fusion protein products (especially NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate or act on downstream targets in a NHP-mediated pathway) can be used to directly treat diseases or disorders. For instance, the administration of an effective amount of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody (or its Fab) that mimics the NHP could activate or effectively antagonize NHP function. Nucleotide constructs encoding such NHP products can be used to genetically engineer host cells to express such products in vivo; these genetically engineered cells function as xe2x80x9cbioreactorsxe2x80x9d in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotide constructs encoding functional NHPs, mutant NHPs, as well as antisense and ribozyme molecules can also be used in xe2x80x9cgene therapyxe2x80x9d approaches for the modulation of NHP expression. Thus, the invention also encompasses pharmaceutical formulations and methods for treating biological disorders.
Various aspects of the invention are described in greater detail in the subsections below.
The cDNA sequences and the corresponding deduced amino acid sequences of the described NHPs are presented in the Sequence Listing. The NHP sequences were obtained from a human prostate cDNA library using probes and/or primers generated from human gene trapped sequence tags. Expression analysis has provided evidence that the described NHPs can be expressed, for example, in a variety of human cell types and that the described NHPs share significant similarity to a variety of proteases, and especially carboxypeptidase B or carboxypetidase A, from, inter alia, humans, mice, and rats. SEQ ID NO: 13 describes a full length NHP ORF with flanking 5xe2x80x2 and 3xe2x80x2 sequences.
NHPs, polypeptides, peptide fragments, mutated, truncated, or deleted forms of the NHPs, and/or NHP fusion proteins can be prepared for a variety of uses. These uses include, but are not limited to, the generation of antibodies, as reagents in diagnostic assays, for the identification of other cellular gene products related to a NHP, as reagents in assays for screening for compounds that can be as pharmaceutical reagents useful in the therapeutic treatment of mental, biological, or medical disorders and disease. Several uses and applications for plasma carboxypeptidases similar to those described herein are described in U.S. Pat. No. 5,593,674, the disclosure of which is herein incorporated by reference in its entirety.
The Sequence Listing discloses the amino acid sequences encoded by the described NHP polynucleotides. The NHPs have initiator methionines in DNA sequence contexts consistent with a translation initiation site and a hydrophobic signal-like sequence is present near the N-terminal region of the protein. The sequence data presented herein indicate that alternatively spliced forms of the NHPs exist (which may or may not be tissue specific).
The NHP amino acid sequences of the invention include the nucleotide and amino acid sequences presented in the Sequence Listing as well as analogues and derivatives thereof. Further, corresponding NHP homologues from other species are encompassed by the invention. In fact, any NHP protein encoded by the NHP nucleotide sequences described above, are within the scope of the invention, as are any novel polynucleotide sequences encoding all or any novel portion of an amino acid sequence presented in the Sequence Listing. The degenerate nature of the genetic code is well known, and, accordingly, each amino acid presented in the Sequence Listing, is generically representative of the well known nucleic acid xe2x80x9ctripletxe2x80x9d codon, or in many cases codons, that can encode the amino acid. As such, as contemplated herein, the amino acid sequences presented in the Sequence Listing, when taken together with the genetic code (see, for example, Table 4-1 at page 109 of xe2x80x9cMolecular Cell Biologyxe2x80x9d, 1986, J. Darnell et al. eds., Scientific American Books, New York, N.Y., herein incorporated by reference) are generically representative of all the various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences.
The invention also encompasses proteins that are functionally equivalent to the NHPs encoded by the presently described nucleotide sequences as judged by any of a number of criteria, including, but not limited to, the ability to bind and cleave a substrate of a NHP, or the ability to effect an identical or complementary downstream pathway, or a change in cellular metabolism (e.g., proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Such functionally equivalent NHP proteins include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the NHP nucleotide sequences described above, but which result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
A variety of host-expression vector systems can be used to express the NHP nucleotide sequences of the invention. The presently described NHPs are similar to plasma carboxypeptidase B and are likely soluble proteins. Where the NHP peptide or polypeptide to be expressed is a soluble NHP protein, or a NHP peptide derived from a substantially nonhydrophobic domain of a NHP, or a truncated or deleted NHP the peptide or polypeptide can be recovered from the culture, i.e., from the host cell in cases where the NHP peptide or polypeptide is not secreted, or from the culture media in cases where the NHP peptide or polypeptide is secreted by the cells. However, such expression systems also encompass engineered host cells that express a NHP, or functional equivalent, in situ, i.e., anchored in the cell membrane. Purification or enrichment of a NHP from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the NHP, but to assess biological activity, e.g., in drug screening assays.
The expression systems that can be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing NHP nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing NHP sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing NHP nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the NHP product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of or containing NHP, or for raising antibodies to a NHP, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target sequence product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign sequences. The virus grows in Spodoptera frugiperda cells. A NHP coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of NHP coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted sequence is expressed (e.g., see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the NHP nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a NHP product in infected hosts (e.g., See Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted NHP nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire NHP sequence or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a NHP coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the sequence product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the NHP sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the NHP product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the NHP product.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk31, hgprt31 or aprt31 cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O""Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).
Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). In this system, the sequence of interest is subcloned into a vaccinia recombination plasmid such that the sequence""s open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
Antibodies that specifically recognize one or more epitopes of a NHP, or epitopes of conserved variants of a NHP, or peptide fragments of a NHP are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(abxe2x80x2)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Antibodies, and applications are uses thereof, similar to those contemplated herein are described in U.S. Pat. No. 5,474,901 the disclosure of which is herein incorporated by reference in its entirety.
The antibodies of the invention can be used, for example, in the detection of NHP in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of NHP. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, in Section 5.5, for the evaluation of the effect of test compounds on expression and/or activity of a NHP coding sequence product. Additionally, such antibodies can be used in conjunction gene therapy to, for example, evaluate the normal and/or engineered NHP-expressing cells prior to their introduction into the patient. Such antibodies may additionally be used as a method for the inhibition of abnormal NHP activity. Thus, such antibodies may, therefore, be utilized as part of treatment methods.
For the production of antibodies, various host animals may be immunized by injection with the NHP, an NHP peptide (e.g., one corresponding the a functional domain of an NHP), truncated NHP polypeptides (NHP in which one or more domains have been deleted), functional equivalents of the NHP or mutated variant of the NHP. Such host animals may include but are not limited to pigs, rabbits, mice, goats, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund""s adjuvant (complete and incomplete), mineral salts such as aluminum hydroxide or aluminum phosphate, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Alternatively, the immune response could be enhanced by combination and or coupling with molecules such as keyhole limpet hemocyanin, tetanus toxoid, diptheria toxoid, ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.
Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
In addition, techniques developed for the production of xe2x80x9cchimeric antibodiesxe2x80x9d (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Such technologies are described in U.S. Pat. Nos. 6,075,181 and 5,877,397 and their respective disclosures which are herein incorporated by reference in their entirety.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce single chain antibodies against NHP sequence products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to: the F(abxe2x80x2)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
Antibodies to a NHP can, in turn, be utilized to generate anti-idiotype antibodies that xe2x80x9cmimicxe2x80x9d a given NHP, using techniques well known to those skilled in the art. (See, e.g., Greenspan and Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). For example antibodies which bind to a NHP domain and competitively inhibit the binding of NHP to its cognate receptor/ligand can be used to generate anti-idiotypes that xe2x80x9cmimicxe2x80x9d the NHP and, therefore, bind and activate or neutralize a receptor, cofactor, ligand, or binding partner. Such anti-idiotypic antibodies or Fab fragments of such anti-idiotypes can be used in therapeutic regimens involving a NHP mediated pathway.

Human carboxypeptidases and polynucleotides encoding the same


Current approaches for determining and identifying whether a correct length of fiber cable is installed between network elements configured to communicate using specific lengths of cable require manual inspection. Such manual inspection may include the use of markers used for visual inspection of cables matching corresponding network elements. An embodiment of the present invention utilizes identifiers unique to cables of various lengths to determine whether a correct length of cable has been installed. Further, embodiments of the present invention determine whether the unique identifier is received by a network device according to a channel mapping corresponding to a unique physical mapping associated with cables of various lengths. The use of the identifiers and channel mappings enable automatic discovery of lengths of cable that are installed between network devices.

Fiber-optic communication is a method of transmitting information from one place to another by sending pulses of light through an optical fiber. The light forms an electromagnetic carrier wave that is modulated to carry information. Because of its advantages over electrical transmission, optical fibers have largely replaced copper wire communications in core networks.
The process of communicating using fiber-optics involves the following basic steps: creating the optical signal involving the use of a transmitter, relaying the signal along the fiber, ensuring that the signal does not become too distorted or weak, receiving the optical signal, and converting it into an electrical signal.
Some optical paths include a working path and protection path, and having both paths operational is useful for integrity and reliability of a network.

System and method for identifying a length of an installed fiber cable